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PubMed2001

CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes.

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.

B-Lymphocytes↗
PubMed1990

A mechanism of activation of the alternative complement pathway by the classical pathway: protection of C3b from inactivation by covalent attachment to C4b.

In this work we studied the role of the classical pathway complement component C4b in the activation of the alternative pathway. It was found that nascent C3b attaches with high efficiency to C4b and that C3b in C4bC3b complexes is protected from inactivation by factors H and I. Activation of C3 by factors B and D in the presence of Mg2+ ions and excess C4b led to 35% incorporation of nascent C3b into C4bC3b complexes in the fluid phase. In comparison, when human IgG was tested as an acceptor under similar conditions, only 12% of generated C3b was incorporated into IgGC3b complexes. The half-life time of dissociation of C3b from purified C4bC3b complexes was approximately 2.3 h at 37 degrees C. C4b in these complexes protected C3b from inactivation as effectively as any known alternative pathway activator. Thus, C3b bound to C4b was tenfold more stable than free C3b or C3b bound to a nonactivating surface. In comparison, the protection provided by attachment to human IgG was only 67% of that of C4b. The results provide an explanation for observations of alternative pathway recruitment following classical pathway activation and for the stability of the classical pathway C5 convertase on surfaces which do not provide protection for C3b from factors H and I.

Complement C3-C5 Convertases↗
PubMed2000

Detection of complement alternative pathway mRNA and proteins in the Alzheimer's disease brain.

Previous research on complement activation in the Alzheimer's disease (AD) brain has focused almost exclusively on the classical complement pathway. The alternative pathway represents another important arm for complement activation, converging with the classical cascade at the C5 cleavage step. Here, we show that mRNA for a critical alternative pathway component, factor B, is present in AD frontal cortex and that the factor D cleaved split products of factor B, Bb and Ba, are significantly increased, indicating alternative pathway activation. By contrast, the two major inhibitors of alternative pathway activation, factor H and factor I, are present at the level of mRNA and protein but are not significantly upregulated. Immunohistochemical analysis reveals significant positive staining in AD sections for all three components. Taken together with previous reports demonstrating alternative pathway activation by amyloid beta peptide, these findings suggest that conditions conducive to chronic alternative pathway activation may exist in the AD brain.

Aged↗
PubMed1996

Distinct characteristics of initiation of the classical and alternative complement pathways by Candida albicans.

Candida albicans is a potent activator of the complement system. The objective of this study was to characterize factors that influence the kinetics for activation of C3 and binding of C3 fragments to C. albicans. Factors that were examined included the surface properties of the yeast and contributions of the classical and alternative complement pathways. The results showed that incubation of hydrophobic, hydrophilic, or germinating yeast cells in normal human serum (NHS) containing radiolabeled C3 led to immediate accumulation of C3 on all three cell types, although the rate of accumulation of C3 on germinating cells was lower. An examination of the sites for early C3 binding showed that classical pathway initiation led to immediate, synchronous binding over the entire cell surface. A blockade of the classical pathway by absorption of putative classical pathway initiators or by chelation of calcium limited activation to the alternative pathway. Binding of C3 solely via the alternative pathway was characterized by a significant lag in the initial binding kinetics. In the absence of classical pathway initiation, the early cellular sites for C3 binding appeared as random, asynchronous foci of C3 that appeared to expand with time. The factor(s) mediating rapid deposition of C3 that was characteristic of the classical pathway initiation was reciprocally cross-absorbed by hydrophilic and hydrophobic C. albicans but was not removed by absorption of NHS with Saccharomyces cerevisiae, encapsulated Cryptococcus neoformans, or nonencapsulated C. neoformans. Delayed binding of C3 produced by absorption of serum was largely reversed by addition to the absorbed serum of immunoglobulin G isolated from NHS, indicating a significant role for a naturally occurring anti-C. albicans immunoglobulin C. in classical pathway initiation.

Antibodies, Fungal↗
PubMed1982

Activation of the alternative complement pathway in ascitic fluid during spontaneous bacterial peritonitis.

We studied complement and immunoglobulin profiles on the serum and ascitic fluid of a patient before and during gram-negative spontaneous bacterial peritonitis (SBP). During the infection, activation of the alternative complement pathway in ascitic fluid was manifested by a 35% reduction in functional activity and depression of both properdin and factor B concentrations to nondetectable levels. Activation of the complement cascade was also demonstrated by a 50% reduction in the C3 concentration and depression of total hemolytic complement. There was no evidence of complement activation of a functionally intact complement system in the ascitic fluid of cirrhotic patients. Complement consumption in ascitic fluid may predispose the cirrhotic to SBP.

Ascitic Fluid↗
PubMed2006

Complement regulation in human atherosclerotic coronary lesions. Immunohistochemical evidence that C4b-binding protein negatively regulates the classical complement pathway, and that C5b-9 is formed via the alternative complement pathway.

OBJECTIVE: The complement system is activated in human atherosclerotic lesions and may hence aggravate local inflammation. We studied the presence and localization of C4b-binding protein (C4bp), the major inhibitor of the classical complement pathway, in human atherosclerotic lesions in relation to complement activation products and protein S, which circulates in complex with C4bp. METHODS AND RESULTS: Immunohistochemistry of human coronary arteries showed C4bp to be virtually absent in normal arteries but present in early and advanced atherosclerotic lesions. In the lesions, C4bp is associated with proteoglycans, and affinity chromatography showed that C4bp interacts with human arterial proteoglycans. Areas containing C4bp also contained IgM and C4 suggesting that C4bp is involved in the regulation of the classical complement pathway. However, C5b-9 was virtually absent in these areas but, instead, colocalized with properdin deeper in the intima, suggesting that C5b-9 is formed by the alternative complement pathway. A fraction of C4bp was associated with protein S and apoptotic cells. CONCLUSIONS: The results indicate that C4bp regulates the classical complement pathway in human atherosclerotic lesions. Thus, unlike the alternative pathway, the classical complement pathway does not generate C5b-9, but is likely to be involved in the clean-up of apoptotic cells and cell debris in the arterial intima.

Apoptosis↗
PubMed1992

Activation of the alternative complement pathway accompanies disease flares in systemic lupus erythematosus during pregnancy.

OBJECTIVE: To assess the activity of systemic lupus erythematosus (SLE) during pregnancy and to distinguish it from preeclampsia. METHODS: We prospectively measured the complement activation products Ba, Bb, SC5b-9, and C4d, as well as the conventional complement determinants C3, C4, and CH50, during pregnancy in 14 patients with SLE and 10 women with preeclampsia. RESULTS: Four of the 14 SLE patients were considered to have disease flares, 3 occurring in the second trimester and 1 postpartum. In these patients, significant abnormalities of Ba, Bb, SC5b-9, and CH50 were noted. In contrast, measures of C4d did not distinguish between pregnant patients who had flares and those whose SLE remained stable. Although decreased values of C3 were rarely seen in the patients with stable disease, normal values of C3 during lupus pregnancy were not reliably associated with stable disease. Three of 10 non-SLE patients with preeclampsia had elevated levels of Ba; however, in each case, the CH50 level was close to or within the normal range. This was in sharp contrast to the findings observed in the 4 patients with active SLE, in whom high levels of plasma Ba were always associated with low CH50 values. Moreover, the ratio of CH50 to Ba was significantly lower in the patients with lupus flares than in the non-SLE patients with preeclampsia. CONCLUSION: While a decline in the CH50 level alone could otherwise be attributed to decreased synthesis of complement components, these data demonstrate that ongoing activation of the alternative complement pathway can accompany disease flares in pregnant women with SLE.

Complement Activation↗
PubMedDate not supplied

Alternative complement pathway in hypocomplementemic/normal C1s-C1 inhibitor complex patients with SLE.

To test whether alternative complement pathway activation explains normal C1s-C1 inhibitor complex in hypocomplementemic (low CH50cl) patients with systemic lupus erythematosus, we examined alternative pathway hemolytic complement (CH50alt) factor B, and Ba fragment in hypocomplementemic sera with normal and with elevated C1s-C1 inhibitor complex. Sera with and without high C1s-C1 inhibitor complex were similar in CH50cl, C3, and C4. There was little evidence for important alternative complement pathway activation in either group, but patients with classical pathway activation (elevated C1s-C1 inhibitor complex) had slightly lower CH50alt and slightly higher factor B and Ba compared to patients with normal C1s-C1 inhibitor complex. Pregnant patients did not differ from non-pregnant patients. Alternative complement pathway activation does not account for hypocomplementemia in this group of patients.

Complement Activation↗
PubMed1985

Immune complexes and complement abnormalities in patients with cystic fibrosis. Increased mortality associated with circulating immune complexes and decreased function of the alternative complement pathway.

Serum samples from 139 patients with cystic fibrosis (CF) were tested for complement abnormalities and circulating immune complexes (CIC). We found no consistent changes in whole complement activity. However, we found CIC in 29% of these patients and decreased activity of the alternative complement pathway (ACP) in 36%. During 5 yr of observation, mortality was much higher in patients whose sera contained CIC (p less than 0.001) or decreased ACP activity (p less than 0.01). Of patients with both abnormalities, 31% died; however, no deaths occurred in patients with normal ACP activity and negative tests for CIC (p less than 0.001). During a subsequent 2.5-yr period, 55% of patients greater than or equal to 21 yr old with both findings died. In contrast, no deaths occurred in older patients lacking this combination (p = 0.0062). Circulating immune complexes but not decreased ACP activity were an independent risk factor for death. Our findings support the hypothesis that humoral immune mechanisms may contribute to morbidity and mortality in CF.

Adolescent↗
PubMed1995

Study of the in vitro activation of the complement alternative pathway by Echinococcus granulosus hydatid cyst fluid.

In the present study we have investigated the fluid phase activation of the complement (C) alternative pathway by Echinococcus granulosus sheep hydatid cyst fluid (SHCF) and its higher molecular weight fraction (SHCF-I) by quantitating the formation of both the terminal C intermediary C5b6 complex and the terminal C complex (TCC). Our results show that in vitro C activation progresses beyond the C5 step suggesting that potentially lytic complexes may be generated in vivo. In addition, SHCF and SHCF-I glucidic moieties are probably involved in C activation since 80% and 86% of SHCF and SHCF-I activity respectively was destroyed by periodate oxidation. Furthermore, partial deglycosylation with Peptide N-Glycosidase F of SHCF-I which had been digested with Pronase E, released an active fraction (MW < 14 KDa) which bound to Soybean agglutinin, suggesting that N-linked oligosaccharides containing alpha- or beta-linked N-acetyl galactosamine play a role in C activation by SHCF.

Acetylgalactosamine↗
PubMed1994

Activation of the alternative complement pathway by red blood cells from patients with sickle cell disease.

Increased activation of the alternative pathway of complement in serum from sickle cell disease (SCD) patients has been reported. We now show that this selective activation is not an artifact of clotting by measuring increased plasma concentrations of Bb in sickle cell patients compared to controls. Furthermore, we show that red blood cells (RBC) from SCD patients activate the alternative complement pathway more than control RBC in an in vitro system. RBC were incubated in 50% normal human serum chelated with 3.5 mM MgCl2 and 10 mM EGTA to block activation by the classical pathway, but allow alternative pathway activation. SCD RBC yielded significantly more activation, as measured by an EIA for C3, P complexes, than control RBC. Denser SCD RBC produced more activation than control RBC, unfractionated SCD RBC, and less dense SCD RBC. These findings are consistent with the hypothesis that dense irreversibly sickled cells, or membrane spicules or vesicles derived from them, may result in complement activation.

Anemia, Sickle Cell↗
PubMed1998

Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway.

Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.

Arginine↗
PubMed1991

Plasma levels of proteins of the alternative complement pathway in inbred mice that differ in resistance to Trypanosoma congolense infections.

Inbred BALB/c, A/J, and C57B1/6J mice were infected with Trypanosoma congolense (Trans Mara strain), clone TC13, and monitored for parasitemia, survival times, and plasma levels of complement components C3, C5, factor B, and factor H. Parasitemia was highest in BALB/c, intermediate in A/J, and lowest in C57Bl/6J mice. The mean survival times were 11.5 +/- 0.9, 23.8 +/- 2.3, and 119 +/- 26 days for BALB/c, A/J, and C57Bl/6J mice, respectively. Preinfection levels of factor H were significantly correlated with survival times (r = 0.7722, P less than 0.001). Marked differences were observed between the plasma levels of C3, factor B, and factor H in the 3 mouse strains following infection. Complement C5 levels showed the fewest changes. In the initial postinfection period, BALB/c mice had highest increases in the levels of the 4 complement proteins but also had the greatest declines toward the end of the infection. Factor H levels showed a biphasic increase in BALB/c and C57Bl/6J, but not in A/J mice, with peaks at days 3 and 9. Complement C3 levels declined in all mice toward the terminal stage of the disease. In the late stages of infection, factor B levels markedly decreased in BALB/c but significantly increased in C57Bl/6J mice. Factor B levels measured at the terminal stages in BALB/c, A/J, and C57Bl/6J were correlated positively with their respective survival times (r = 0.714, P less than 0.01). The results suggest that genetic differences in the alternative complement pathway might affect the resistance to T. congolense infections.

Animals↗
PubMed1996

Selective inhibition of the alternative complement pathway by sCR1[desLHR-A] protects the rabbit isolated heart from human complement-mediated damage.

Evidence is presented that treatment with a selective inhibitor of the alternative complement pathway, sCR1[desLHR-A], protects the ex vivo perfused rabbit heart from human complement-mediated injury. Hearts from male New Zealand white rabbits were perfused in the Langendorff mode. After equilibration, normal human plasma was added to the perfusate as a source of complement. Concomitant with the addition of human plasma, vehicle (n = 13), soluble complement receptor type 1 (sCR1) (n = 10), or sCR1[desLHR-A], a truncated version of sCR1 that lacks the C4b binding region (n = 10) was included in the perfusate. Hemodynamic variables were obtained for all groups before (baseline) and after the addition of human plasma. Compared to vehicle-treated hearts, variables recorded during perfusion with human plasma including coronary perfusion pressure, left ventricular developed pressure, and left ventricular end diastolic pressure, along with a reduction of creatine kinase efflux, were improved in hearts perfused with either complement inhibitor. In addition, in vitro hemolysis assays were utilized to discriminate between the classical and alternative pathways. The addition of sCR1 to human serum prevented both the classical and alternative pathway-mediated hemolysis while sCR1[desLHR-A] prevented only the alternative pathway-mediated lysis. This study indicates that deletion of the C4b-binding site from sCR1 results in a new pharmacological moiety, sCR1[desLHR-A], that primarily inhibits the alternative pathway of human complement.

Animals↗
PubMed1988

Human peritoneal macrophages. Production in vitro of the active terminal complement components C5 to C9 and a functional alternative pathway of complement. Brief report.

Endotoxin-stimulated human peritoneal macrophages were cultured in serum-free medium with agarose beads. Monospecific antibodies to human C3c, C3g, C5, C6, C7, C8, C9 and to C9-neoantigen bound to the beads. This shows that activated C3 and the terminal complement complex (TCC), made from complement components C5 to C9, were generated on the beads. De novo synthesis was confirmed by agarose binding of tritium-labelled protein. Moreover, C3-derivatives and C9-neoantigen were detected on normal serum-treated agarose beads but not on beads kept in factor B-depleted or heat-inactivated sera, implying that an intact alternative complement pathway was required for our findings. The macrophages thus synthesize the active complement components of the alternative and terminal pathways in vitro.

Ascitic Fluid↗
PubMed1997

Experimental murine acid aspiration injury is mediated by neutrophils and the alternative complement pathway.

Acid aspiration may result in the development of the acute respiratory distress syndrome, an event associated with significant morbidity and mortality. Although once attributed to direct distal airway injury, the pulmonary failure after acid aspiration is more complex and involves an inflammatory injury mediated by complement (C) and polymorphonuclear leukocytes. This study examines the injurious inflammatory cascades that are activated after acid aspiration. The role of neutrophils was defined by immunodepletion before aspiration, which reduced injury by 59%. The injury was not modified in either P- or E-selectin-knockout mice, indicating that these adhesion molecules were not operative. C activation after aspiration was documented with immunochemistry by C3 deposition on injured alveolar pneumocytes. Animals in which C activation was inhibited with soluble C receptor type 1 (sCR1) had a 54% reduction in injury, similar to the level of protection seen in C3-knockout mice (58%). However C4-knockout mice were not protected from injury, indicating that C activation is mediated by the alternative pathway. Finally, an additive effect of neutrophils and C was demonstrated whereby neutropenic animals that were treated with sCR1 showed an 85% reduction in injury. Thus acid aspiration injury is mediated by neutrophils and the alternative C pathway.

Animals↗
PubMed1996

Key role of the alternate complement pathway in hyperacute rejection of rat hearts transplanted into fetal sheep.

Hyperacute rejection (HAR) mediated by xenoreactive natural antibodies (XNA), which are thought to develop in early infancy, is a major impediment to transplantation between widely disparate species. The ability to diagnose certain forms of congenital heart defects early in prenatal life suggests the potential for these defects to be corrected by cardiac transplantation, prior to the development of XNA and host immunocompetence. This study investigated whether discordant cardiac xenotransplantation into fetal and neonatal recipients might obviate HAR due to the relative lack of XNA. Six neonatal lambs at 3 days (n=3) or 7 days (n=3) of life, and two fetal lambs at 125 and 142 days of gestation (term = 145 days) received cardiac grafts from adult Wistar-Furth (275-350 g) rats. All eight cardiac xenografts showed clinical evidence of HAR, with rapid swelling, loss of contractility, and ecchymosis and a mean survival time of 12.5 +/- 6.4 min. Sections of explanted grafts showed classical histologic features of HAR, including interstitial hemorrhages, platelet microthrombi, and edema, without leukocyte infiltration. Immunopathology of grafts harvested from fetal recipients showed a lack of significant intragraft deposition of sheep IgM, IgG, or C1q, but widespread endothelial labeling for C3, factor B, and properdin. In contrast, grafts in neonatal recipients showed IgM, IgG, and C1q deposition, as well as C3, factor B, properdin, and terminal complement (C) components. Fibrin deposition and platelet thrombi were seen in both groups of recipients. Injection of cobra venom factor resulted in prolongation of cardiac xenograft survival in neonatal lambs (n=3) to 12 hr. Analysis by immunohistology showed that normal sera from neonatal and adult, but not fetal, sheep contained IgM and IgG XNA reactive with rat cells. In conclusion, rodent grafts transplanted into fetal sheep undergo HAR, likely through direct activation of the alternate pathway of C, whereas neonatal lambs acquire XNA in the very early postnatal period and reject rat hearts through activation of both the classical and alternate pathways of C. Thus, at least in some species combinations, cardiac transplantation during the early postnatal period, or even in utero, may still be subject to development of HAR.

Acute Disease↗